This proposal describes a 5-year career development program designed to support an academic, physician- scientist career. The proposed research project will capitalize on the expertise and resources available at Washington University, which has a proven track record of developing physician-scientists. Dr. Timothy Ley, an expert in myeloid development and cancer genomics, and a recipient of the American Society of Hematology Mentor Award, will serve as the research mentor. This award will enable the candidate to obtain practical and/or didactic training in scientific methodology and technical skills for the purpose of establishing an independent research program focused on gene regulation in hematopoietic development and cancer biology. The long-term goal of this project is to understand the regulatory mechanisms controlling HOX gene expression in normal hematopoiesis and acute myeloid leukemia (AML). The HOX genes have established roles in self-renewal in normal hematopoiesis and are highly expressed in the majority of AML patients. Although specific AML mutations are associated with canonical patterns of HOX gene expression, the regulatory pathways responsible for these patterns are unknown. The proposed research will use in vitro hematopoietic differentiation of human embryonic stem cells (ESCs) and primary human AML samples to study the regulation of HOX genes in normal human hematopoiesis and AML. The objectives of this project are to identify the key cis-acting DNA elements and upstream transcription factors involved in HOX gene regulation during normal hematopoiesis, and to investigate the changes in the HOX gene expression program that are caused by specific AML mutations. These objectives will be pursued in the following Specific Aims: Specific Aim 1: We will define the regulatory mechanisms controlling HOX gene expression during normal human hematopoietic development. HOX gene expression and chromatin structure will be assessed in human ESCs during in vitro hematopoietic differentiation, and the regulatory DNA elements will be functionally validated using chromatin immunoprecipitation and other targeted approaches; and Specific Aim 2: We will determine the impact of AML mutations associated with canonical HOX phenotypes on the HOX expression program in hematopoietic progenitors and primary AML samples. The HOX expression and regulatory landscape will be analyzed in human ESCs that express the MLL-ENL, CBFB-MYH11, NPMc mutations. Mutation-specific changes will be investigated using targeted methods, and findings from this model system will be verified using primary samples from AML patients. These studies will define new mechanisms of HOX gene regulation in AML cells, and may yield novel approaches for the treatment of this malignancy. Importantly, the proposed research also establishes a model system for studying the regulation of HOX and other genes in early hematopoietic stem/progenitor cells, which will provide the applicant with the foundation for an independent research program.